Claude Lhuillery

Enhancement of doxorubicin cytotoxicity by polyunsaturated fatty acids in the human breast tumor cell line MBA‐MB‐231: Relationship to lipid peroxidation

Exogenous polyunsaturated fatty acids modulate the cytotoxic activity of anti‐cancer drugs. In this study, we examined whether lipid peroxidation is a potential mechanism through which fatty acids enhance drug cytotoxicity. We measured cell viability in the human breast cancer cell line MDA‐MB‐231 exposed to doxorubicin in the presence of non‐cytotoxic concentrations of various polyunsaturated fatty acids for 6 days. To determine the role of lipid peroxidation, the hydroperoxide level was measured in cell extracts. Among all polyunsaturated fatty acids tested, docosahexaenoic acid (DHA, 22:6n‐3) was the most potent in increasing doxorubicin cytotoxicity: cell viability decreased from 54% in the presence of 10−7 M doxorubicin alone to 21% when cells were incubated with doxorubicin and DHA. After addition of an oxidant system (sodium ascorbate/2‐methyl‐1,4‐naphthoquinone) to cells incubated with doxorubicin and DHA, cell viability further decreased to 12%. Cell hydroperoxides increased commensurately. The effect of DHA on doxorubicin activity and lipid hydroperoxide formation was abolished by a lipid peroxidation inhibitor (dl‐α‐tocopherol) or when oleic acid (a non‐peroxidizable fatty acid) was used in place of DHA. No effect was observed with mitoxantrone, a drug with a low peroxidation‐generating potential. Thus, DHA may increase the efficacy of oxyradical‐producing drugs through a mechanism involving a generation of lipoperoxides. This may lead in vivo to a modulation of tumor cell chemosensitivity by DHA and oxidant agents. Int. J. Cancer 75:578–583, 1998.

Low alpha-linolenic acid content of adipose breast tissue is associated with an increased risk of breast cancer

Data derived from experimental studies suggest that alpha-linolenic acid may have a protective effect in breast cancer. Observations obtained from epidemiological studies have not allowed conclusions to be drawn about a potential protective effect of dietary alpha-linolenic acid on breast cancer, possibly because of methodological issues. This case-control study conducted in an homogeneous population from a central area in France was designed to explore the hypothesis that alpha-linolenic acid inhibits breast cancer, using fatty acid levels in adipose breast tissue as a biomarker of past qualitative dietary intake of fatty acids. Biopsies of adipose breast tissue at the time of diagnosis were obtained from 123 women with invasive non-metastatic breast carcinoma. 59 women with benign breast disease served as controls. Individual fatty acids were analysed by capillary gas chromatography. An unconditional logistic regression model was used to obtain odds ratio estimates whilst adjusting for age, menopausal status and body mass index (BMI). No association was found between fatty acids (saturates, monounsaturates, long-chain polyunsaturates n-6 or n-3) and the disease, except for alpha-linolenic acid which showed an inverse association with the risk of breast cancer. The relative risk of breast cancer for women in the highest quartile of adipose breast tissue alpha-linolenic acid level was 0.36 (95% confidence interval=0.12-1.02) compared with those in the lowest quartile (P trend=0.026), suggesting a protective effect of alpha-linolenic acid in the risk of breast cancer. The effects of dietary alpha-linolenic on the risk of breast cancer warrant further study.

Effect of an a-Linolenic Acid-Rich Diet on Rat Mammary Tumor Growth Depends on the Dietary Oxidative Status

To investigate whether the oxidative status of an 18:3(n-3) polyunsaturated fatty acid (PUFA)-enriched diet could modulate the growth of chemically induced rat mammary tumors, three independent experiments were performed. Experiments I and II examined the variation of tumor growth by addition of antioxidant (vitamin E) or a prooxidant system (sodium ascorbate/2-methyl-1,4-naphthoquinone) to a 15% linseed oil diet rich in 18:3(n-3). Experiment III addressed the role of PUFA in the tumor growth modulation by vitamin E. For this purpose, we compared the effect of vitamin E in 15% fat diets containing a high level of 18:3(n-3) (linseed oil, high-PUFA diet) or devoid of 18:3(n-3) (hydrogenated palm/sunflower oil, low-PUFA diet). In Experiments I-III, tumor growth increased in the presence of vitamin E compared with control (without vitamin E). Furthermore, it decreased when prooxidant was added. In contrast, no difference was observed when the diet was low in PUFA, suggesting that sensitivity of PUFA to peroxidation may interfere with tumor growth. This observation was supported by growth kinetic parameter analysis, which indicated that tumor growth resulted from variations in cell loss but not from changes in cell proliferation. These data show that, in vivo, PUFA effects on tumor growth are highly dependent on diet oxidative status.

Dietary n-3 polyunsaturated fatty acids and oxidants increase rat mammary tumor sensitivity to epirubicin without change in cardiac toxicity.

We previously reported that polyunsaturated fatty acids (PUFA) and oxidants (OX) increased in vitro efficacy of anticancer drugs that generate reactive oxygen species, through an increase in lipid peroxides formation (1). The aim of the study was to examine whether n-3 PUFA and OX modulate in vivo the antitumor activity and cardiotoxicity of epirubicin, an anthracyclin that stimulates oxidative stress. Thirty-six rats with N-methyl nitrosourea-induced mammary tumors were fed a basal diet supplemented with n-3 PUFA and separated in three groups receiving n-3 PUFA alone (15% sardine oil, control group) or with lipid peroxidation inhibitor (vitamin E, 100 lUNg, vit E group) or with lipid peroxidation inducers (dehydroascorbate + menadione, respectively, 20 and 0.2 rag/d, OX group). When the target tumor reached 1 cm in diameter, epirubicin (3 mg/kg; i.p.) was administered weekly for 3 wk. Tumor response was assessed in two ways: the change in tumor area and the score of new tumors occurring after chemotherapy. Four weeks after the end of treatment, cardiac functioning was evaluated by measuring the left ventricular end-diastolic pressure at basal conditions or during volume expansion. During a 6-wk period, the growth of the target tumor was totally inhibited in the OX group by the epirubicin treatment, while it kept going in the control and in the vit E groups. Similarly, the number of newly appeared tumors was reduced three times in the OX group in comparison to both the other groups. No difference in the cardiac pressures measured at rest or during volume expansion was observed between the dietary groups. However, almost 10% of rats in all dietary groups were found to present an elevated left ventricular enddiastolic pressure, which is characteristic of the cardiotoxicity induced by anthracyclin. It is concluded that dietary n-3 PUFA in the presence of OX increased the antitumor activity of epirubicin in vivo, without affecting cardiac toxicity. This observation suggests that supplementing the diet with n-3 PUFA plus OX increases the cytotoxicity of epirubicin toward tumor cells rather than toward normal cardiac cells. A phase I-II is now contemplated in metastatic breast cancer patients in Tours.

Dietary purified cis-9,trans-11 conjugated linoleic acid isomer has anticarcinogenic properties in chemically induced mammary tumors in rats

To determine whether the purified 9c,11t conjugated linoleic acid (CLA) isomer, the main dietary isomer, is biologically active on mammary tumor growth, we carried out a dietary intervention study designed to compare its effects with those of a mixture of CLA isomers on the incidence and growth of autochthonous mammary tumors induced by methylnitrosourea in rats. After the initiation step, rats were fed a sunflower oil-based diet (5%) and separated into three experimental groups supplemented with either a 1% homemade synthesized 9c,11t isomer, a 1% CLA isomer mixture, or free fatty acids prepared from sunflower oil for the control group. We found that, in the two CLA groups compared with the control group, CLA levels were about 30 times higher in mammary fat pads and about 10 times higher in tumor tissues. Compared with the control group, there was a 44% and 45% decrease in tumor mass per rat in the CLA mixture and the 9c,11t groups, respectively, at 20 wk of diet (P < 0.05). There was a nonsignificant trend for a decrease multiplicity in CLA groups compared with the control group, with a 30% and 35% decrease in the CLA mixture and the 9c,11t groups, respectively. Incidence and latency were not significantly different between the dietary groups. Although the effect was specifically restricted in reduction in tumor mass, we concluded that the main CLA isomer found in human diet has anticarcinogenic properties in experimental mammary carcinogenesis.

Suppression of the promoter effect of polyunsaturated fatty acids by the absence of dietary vitamin E in experimental mammary carcinoma

Because lipoperoxides seem able to modulate tumor growth, we have examined the concomitant effects of dietary antioxidant (vitamin E) and of a high amount of polyunsaturated fatty acids (PUFA) in a model of chemically-induced mammary carcinogenesis. Two groups of rats received a high fat diet with or without added vitamin E and mammary tumors were initiated in both groups by an injection of carcinogen. Tumor growth was followed in both groups. We found that tumor incidence and growth were decreased in rats with no added vitamin E in diet, suggesting a protective role of oxidized PUFA at later stages of carcinogenesis.

Time-course study of adipose tissue fatty acid composition during mammary tumor growth in rats with controlled fat intake

Previous work in breast cancer patients has indicated an inverse relationship between the risk of relapse and the alpha-linolenic acid (18:3n-3) level in adipose breast tissue. To determine whether low alpha-linolenic levels in patients with aggressive breast cancer resulted from lower 18:3n-3 dietary intake and/or increased metabolism of stored 18:3n-3, we analyzed the fatty acid composition of mammary adipose tissue during tumor growth in a rat model of mammary carcinogenesis. Rats were fed a diet containing 10% fat as rapeseed oil (in which 9% of total fatty acids is 18:3n-3). one-half of the rats received an injection of nitrosomethylurea (NMU) to initiate mammary tumors. In control and NMU-treated groups, three to five animals were sacrificed every three weeks during the five-month experimental time. tumor growth was followed by weekly palpation of the animals and by the measure of total tumor mass and number in sacrificed rats. Mammary tumor and adipose tissues were sampled in sacrificed rats. We found that although mammary adipose tissue fatty acid profile changed throughout the experiment, there was no difference in fatty acid profile between control and NMU-treated rats of the same age. In the NMU-treated group, 18:3n-3 level remained identical throughout the experimental period, irrespective of tumor burden. These data show that, in this model, mammary tumor growth does not modify stored fatty acid levels, including 18:3n-3. this suggests that decreased 18:3n-3 level in patients with poor prognosis is not a consequence of tumor burden but more likely depends on decreased dietary intake.

Alpha‐tocopherol and hydroperoxide content in breast adipose tissue from patients with breast tumors

The study of the relationship between dietary intake of vitamin E and the risk of breast cancer has not yielded definite conclusions with respect to causality, possibly due to methodological issues inherent to nutritional epidemiology. To avoid the pitfalls of dietary recalls, α‐tocopherol content of adipose tissue was used as a biochemical indicator of long‐term dietary intake of vitamin E. α‐tocopherol and hydroperoxides were measured in breast adipose tissue obtained at the time of diagnosis from 70 patients with early breast cancer. Thirty women with non‐malignant breast tumors served as control. Lipid peroxidation was monitored by quantifying conjugated dienes spectrophotometrically and by assaying hydroperoxides with an iodometric method; α‐tocopherol was measured by HPLC associated with fluorescence detection. Mean α‐tocopherol value in breast adipose tissue was significantly lower in breast cancer patients than in control patients, whereas the hydroperoxide content was significantly higher in cancer patients than in controls. The α‐tocopherol concentration in adipose tissue was not correlated with the clinical status of the patients with respect to age, menopausal status or body mass index. We conclude from this pilot study that breast cancer is associated with a low content of α‐tocopherol in breast adipose tissue, and with an altered lipid oxidation pattern, which might be related to a low antioxidant status.

Effects of dietary fat and adipose tissue location on insulin action in young boar adipocytes

1. Regulation of lipogenesis and lipolysis by insulin was studied on adipocytes isolated from 100 kg Large white male pigs. Two adipose tissues were studied: subcutaneous and perirenal. Animals were fed either a control low fat diet or a diet containing 14.7% sunflower seed oil. 2. The cell diameter was higher in the group fed the sunflower diet. 3. De novo lipogenesis was decreased for each adipose tissue in the group fed the sunflower diet. The perirenal site had a higher lipogenic activity than subcutaneous site whatever the diet. 4. Insulin did not significantly stimulate lipogenesis but had an important antilipolytic effect on stimulated lipolysis by isoproterenol. 5. The antilipolytic action of insulin was higher in perirenal adipocytes with the control diet. With the sunflower diet, the decrease was about 54.4% for subcutaneous adipocytes, whereas the inhibition was decreased in perirenal adipocytes. Addition of theophylline reversed the antilipolytic action of insulin. 6. Insulin binding was not affected neither by the dietary fat nor by the adipose tissue location. 7. Absence of de novo lipogenesis stimulation by insulin was not due to an impairment in insulin binding. 8. The different effects of dietary fat and adipose tissue location on the antilipolytic action of insulin could not be explained by a modification of insulin binding but rather by a latter event, probably at a post-insulin binding stage.

Comparative study of triacylglycerol fatty acids in milk fat from two leporidae species: rabbit (oryctolagus cuniculus) and hare (Lepus europaeus)

1. Fatty-acid composition of hare milk triacylglycerols markedly differed from that of rabbit milk and was essentially characterized by a 2.1 times lower concentration of medium-chain fatty-acids (M.C.F.A.). 2. Consequently, very high levels (greater than 70 moles %) of M.C.F.A., previously reported for rabbit milk, cannot be considered as a general characteristic of the Leporidae. 3. General tendencies recorded for intramolecular fatty acid distribution in hare milk triacylglycerols agreed with those reported for other animal species.